多药耐药对肝癌细胞增殖、凋亡、侵袭活性、耐药机制及丝裂原活化蛋白激酶表达的影响  

The effects of multidrug resistance on cell proliferation, apoptosis and invasion activity and the expression of mitogen-activated protein kinase in human hepatocellular cancer cells

作　　者：颜昕[1] 阮文雯[1] 王效民[2] 丁鑫[3] 郝兰香[3] 廖洪锋[3] 潘超[3] 

机构地区：[1]厦门大学医学院临床医学系,厦门361003 [2]厦门大学附属中山医院肝胆外科,厦门361004 [3]厦门大学附属中山医院病理科,厦门361004

出　　处：《肿瘤》2012年第7期507-515,共9页Tumor

摘　　要：目的:通过化疗药物诱导建立多株人肝癌多药耐药细胞模型,探讨多药耐药获得对细胞增殖、凋亡、侵袭能力的影响,耐药机制及与丝裂原活化蛋白激酶(mitogen-activated protein kinase,MAPK)信号通路表达的相关性。方法:采用多柔比星(adriamycin,ADM)高浓度冲击和小剂量缓升2种方法诱导建立人肝癌细胞(HepG2、SMMC-7721和BEL-7402)耐药模型。比较耐药细胞株与亲本细胞株间的差异,主要包括采用生物发光法进行药物敏感实验并计算耐药指数;RT-PCR法和免疫组织化学法检测相关耐药基因(P-糖蛋白、多药耐药相关蛋白1、肺癌耐药蛋白、乳腺癌耐药蛋白、蛋白激酶C、谷胱甘肽-S-转移酶-π和拓扑异构酶Ⅱ)mRNA及相应蛋白的表达。免疫组织化学法检测与细胞增殖活性相关的Ki-67和增殖细胞核抗原(proliferating cell nuclear antigen,PCNA)的表达;FCM法检测细胞周期、细胞凋亡率的改变;Transwell小室法检测细胞体外侵袭能力的改变;最后,分别采用RT-PCR和蛋白质印迹法检测MAPK信号通路中ERK1、ERK2、ERK5、JNK1、JNK2和p38α基因及相应蛋白的表达。结果:共建立6株ADM耐药细胞株;与亲本细胞株相比,耐药细胞株耐药指数均>5,并对多种化疗药耐受性增强,多药耐药相关基因及蛋白的表达均上调2～10倍,以高浓度冲击法诱导的细胞中蛋白表达上调明显。耐药细胞中Ki-67及PCNA蛋白的表达量均升高20倍以上,细胞周期被阻滞在S期,体外侵袭能力增强1.3～2.5倍;ADM干预后,细胞凋亡率减少60%以上。MAPK信号通路中不同基因和蛋白表达量均有不同比例升高,以ERK1、ERK2升高明显。结论:ADM能够诱导多株人肝癌细胞产生多药耐药,导致细胞增殖活性升高、细胞周期改变、抗凋亡能力及侵袭能力增强。MAPK信号通路中相关蛋白表达的上调与细胞多药耐药有一定关系。Objective： To establish multiple human hepatocellular carcinoma （HCC） multidrug-resistant cell lines induced by chemotherapeutic drugs, and to investigate the effects of multidrug-resistance on cell proliferation, apoptosis and invasion activity and the relationship between the multidrug-resistance and the expression of mitogen-activated protein kinase （MAPK） pathway. Methods： Three different human HCC cell strains HepG2, SMMC-7721 and BEL-7402 were established by using pulse treatment with high concentration of adriamycin （ADM） or treatment with ADM of low concentration gradually increased. The difference between the resistant cells and the parental cells was evaluated. The drug sensitivity was tested by ATP bioluminescence to calculate the resistance index （RI）. The expressions of multidrug resistance-related genes including P-glycoprotein （P-gp）, multidrug resistance-associated protein I （MRPI）, lung cancer resistance protein 1 （LRPI）, breast cancer resistance protein （BCRP）, glutathione-S- transferase-π （GST-π）, topoisomerase Ⅱ （ToPo 113） and protein kinase C （PKC） and their proteins were detected by RT-PCR and immunohistochemistry （IHC）, respectively. The expressions of cell proliferative activity-related Ki-67 and proliferating cell nuclear antigen （PCNA） were detected by IHC. The cell cycle and apoptosis rate were detected by flow cytometry （FCM）, and the change of cell invasion ability was detected by Transwell assay. The expressions of ERK1, ERK2, ERK5,JNK1 ,JNK2 and p38a genes and their proteins in MAPK pathway were detected by RT-PCR and Western boltting, respectively. Results： Six ADM- resistant cell strains were established. Compared to parental cells, the RIs of all resistant cells were over 5 with increased resistance to multiple chemotherapeutic drugs. The expressions of MDR genes and their proteins were increased 2-10 times, especially for the cells induced by pulse treatment with high concentration of ADM. The expressions of Ki-67

关 键 词：肝肿瘤 多药耐药相关蛋白类 丝裂原激活蛋白激酶类 

分 类 号：R735.7[医药卫生—肿瘤]